DIGE analysis of proteome changes accompanying large resveratrol production by grapevine (Vitis vinifera cv. Gamay) cell cultures in response to methyl-β-cyclodextrin and methyl jasmonate elicitors

We had previously shown that Vitis vinifera cv. Gamay cell suspension accumulates extracellularly large amounts of the phytoalexin trans-resveratrol (tR) in response to elicitation with methylated cyclodextrins (MBCD), which can be triplicated when the elicitor is combined with methyl jasmonate (MeJA). In parallel, new pathogenesis-related proteins accumulated in the apoplast-like extracellular space. The aim of this study was to investigate changes in the grapevine cell proteome potentially related to tR accumulation in response to the above elicitors. The DIGE technique was used to detect statistically significant changes in the cell's proteome. A total number of 1031 unique spots were detected, 67 of which were de-regulated upon elicitation. Sixty-four spots were successfully identified by nLC-MS/MS database search analysis. The tR biosynthetic pathway enzymes were up-regulated by MBCD alone or combined with MeJA, but not by treatment with MeJA alone, in agreement with tR accumulation pattern. Seven spots contained stilbene synthase encoded by four different isogenes. Likewise, four glutathione-S-transferases, potentially involved in tR trafficking within the cell and across membranes, were up-regulated in the same fashion as stilbene synthases. The relation of other de-regulated proteins with other effects caused by elicitors on grapevine cells, namely defense response and cell growth inhibition, is discussed.     

Implication of signaling pathways involving calcium, phosphorylation and active oxygen species in methyl jasmonate-induced defense responses in grapevine cell cultures

Perception of elicitors triggers plant defense responses via various early signal transduction pathways. Methyl jasmonate (MeJA) stimulates defense responses in grapevine (Vitis vinifera). We investigated the involvement of various partners (calcium, ROS, reversible phosphorylation) in MeJA-induced responses by using a pharmacological approach. We used specific calcium channel effectors and inhibitors of serine/threonine phosphatases, superoxide dismutase and NAD(P)H oxidase and investigated production of stilbenes (resveratrol and its glucoside, piceid, the major form), which are the grapevine phytoalexins. RNA accumulation of two genes encoding enzymes involved in stilbene synthesis (PAL and STS), three genes encoding pathogenesis-related proteins (CHIT4C, PIN and GLU) and one gene encoding an enzyme producing jasmonates (LOX) were also assessed. Calcium and its origin seemed to play a major role in MeJA-induced grapevine defense responses. Phytoalexin production was strongly affected if calcium from the influx plasma membrane was inhibited, whereas calcium from the intracellular compartments did not seem to be involved. ROS production seemed to interfere with MeJA-stimulated defense responses, and protein phosphorylation/dephosphorylation events also played a direct role.     

Changes of defense proteins in the extracellular proteome of grapevine (Vitis vinifera cv. Gamay) cell cultures in response to elicitors

In plant cells, elicitors induce defense responses that resemble those triggered by pathogen attack, such as the synthesis of phytoalexins and pathogen-related proteins which accumulate in the extracellular space. In the search for the particular proteins involved in defense responses, we investigated the changes in the extracellular proteome of a grapevine (Vitis vinifera cv. Gamay) cell suspension in response to elicitation with methylated cyclodextrins (MBCD) and methyl jasmonate (MeJA). Twenty-five of the 39 spots differentially expressed in 2-D gels were identified and found to be encoded by 10 different genes: three secretory peroxidases, chitinase-III, β-1,3-glucanase, thaumatin-like, SGNH plant lipase-like, NtPR27-like, xyloglucan endotransglycosylase and subtilisin-like protease. Most of them belong to the pathogenesis-related type proteins. A new class III secretory basic peroxidase and chitinase III were strongly induced in cultures treated with MBCD alone or combined with MeJA, while cultures treated with MeJA alone displayed a general repression of most of the extracellular proteins. Some of the proteins induced in grapevine cell cultures by MBCD are induced in other species by activators of systemic acquired resistance (SAR), a form of plant immunity. Collectively, the results suggest that treatment with MBCD resembles the effect of SAR induction agents in cell cultures.     

Mutation of a family 8 glycosyltransferase gene alters cell wall carbohydrate composition and causes a humidity-sensitive semi-sterile dwarf phenotype in Arabidopsis

Incompatible plant-pathogen interactions result in the rapid cell death response known as hypersensitive response (HR) and activation of host defense related genes. To understand the cellular mechanism controlling defense response better, a novel pathogenesis-related (PR) gene and putative cell wall protein gene, CaTin2, was isolated through differential screening of a hot pepper cDNA library and characterized. CaTin2 gene was locally and systemically induced in hot pepper plants upon TMV-P₀ inoculation which induces HR. However, CaTin2 gene wasn't regulated by bacterial HR-specific signal pathway. The full-length cDNA for CaTin2, which is 864 nucleotides long, contained the open reading frame of 200 amino acids including cell wall targeting sequences of 26 amino acids. CaTin2 gene has no sequence similarity with other cell wall protein genes except the signal sequence and exists as only one copy in hot pepper genome. CaTin2 gene contains repeated helix-turn-helix motif consisting of 39 amino acids. CaTin2 mRNA accumulation was induced in response to various treatments such as ethylene, SA, MeJA, ABA, methyl viologen, NaCl and wounding at early time points. Subcelluar localization of CaTin2 was confirmed in the cell wall in hot pepper leaves by making CaTin2::smGFP fusion protein. The transgenic plants overexpressing CaTin2 cDNA were resistant to TMV and CMV inoculation. From these results, CaTin2 gene may encode a virus-related new cell wall protein member.     

Comparative proteomic analysis of methyl jasmonate‐induced defense responses in different rice cultivars

Jasmonate is an important endogenous chemical signal that plays a role in modulation of plant defense responses. To understand its mechanisms in regulation of rice resistance against the fungal pathogen Magnaporthe oryzae, comparative phenotype and proteomic analyses were undertaken using two near‐isogenic cultivars with different levels of disease resistance. Methyl‐jasmonate (MeJA) treatment significantly enhanced the resistance against M. oryzae in both cultivars but the treated resistant cultivar maintained a higher level of resistance than the same treated susceptible cultivars. Proteomic analysis revealed 26 and 16 MeJA‐modulated proteins in resistant and susceptible cultivars, respectively, and both cultivars shared a common set of 13 proteins. Cumulatively, a total of 29 unique MeJA‐influenced proteins were identified with many of them known to be associated with plant defense response and ROS accumulation. Consistent with the findings of proteomic analysis, MeJA treatment increased ROS accumulation in both cultivars with the resistant cultivar showing higher levels of ROS production and cell membrane damage than the susceptible cultivar. Taken together, our data add a new insight into the mechanisms of overall MeJA‐induced rice defense response and provide a molecular basis of using MeJA to enhance fungal disease resistance in resistant and susceptible rice cultivars.     

Characterization of Plasmopara-resistance in grapevine using in vitro plants

Although the exact mechanisms by which grapevine cells operate to reduce disease incidence caused by the downy mildew fungus Plasmopara viticola are not fully elucidated, our cytological results obtained from infected in vitro-plants confirm that enhanced disease resistance is associated with an expression of distinct reactions in a chronological order. An increased production of reactive oxygen species (superoxide radicals, 4-6 hours post infection, hpi) was followed by a hypersensitive response (6-8 hpi), an increased activity of peroxidase in cells flanking the infection area and in the vascular tissue (10-12 hpi) and an increased production, accumulation or conversion of phenolic compounds (12-15 hpi). These mechanisms seem also to be present in susceptible varieties as shown after an inoculation with non-host oomycetic pathogens on the basis of peroxidase activity, but they do not become activated after P. viticola infection. The investigation of the peroxidase activity in leaves at several time points after an infection with P. viticola indicated that there is a strong correlation between the POX activity in leaves of in vitro-plants and the resistance of grapevine plants to P. viticola in the field.     

Proteomics of Arabidopsis redox proteins in response to methyl jasmonate

Protein redox regulation is increasingly recognized as an important switch of protein activity in yeast, bacteria, mammals and plants. In this study, we identified proteins with potential thiol switches involved in jasmonate signaling, which is essential for plant defense. Methyl jasmonate (MeJA) treatment led to enhanced production of hydrogen peroxide in Arabidopsis leaves and roots, indicating in vivo oxidative stress. With monobromobimane (mBBr) labeling to capture oxidized sulfhydryl groups and 2D gel separation, a total of 35 protein spots that displayed significant redox and/or total protein expression changes were isolated. Using LC–MS/MS, the proteins in 33 spots were identified in both control and MeJA-treated samples. By comparative analysis of mBBr and SyproRuby gel images, we were able to determine many proteins that were redox responsive and proteins that displayed abundance changes in response to MeJA. Interestingly, stress and defense proteins constitute a large group that responded to MeJA. In addition, many cysteine residues involved in the disulfide dynamics were mapped based on tandem MS data. Identification of redox proteins and their cysteine residues involved in the redox regulation allows for a deeper understanding of the jasmonate signaling networks.     

The effects of herbicides and mycoparasites at different moisture levels on carpogenic germination in Sclerotinia sclerotiorum

Colonization of plant roots by fluorescent pseudomonads has been correlated with disease suppression. One mechanism may involve altered defense responses in the plant upon colonization. Altered defense responses were observed in bean (Phaseolus vulgaris) inoculated with fluorescent pseudomonads. Systemic effects of root inoculation by Pseudomonas putida isolate Corvallis, P. tolaasii (P9A) and P. aureofaciens REW1-I-1 were observed in bean leaves from 14-day-old plants. SDS- polyacrylamide gel electrophoresis demonstrated that levels of certain acid-soluble proteins increased in the leaf extracts of inoculated plants. Plants inoculated with REW1-I-1 produced more of a 57 M_r protein, and plants inoculated with isolates P9A and REW1-I-1 produced more of a 38 M_r protein. Northern hybridization revealed enhanced accumulation of mRNAs, that encode the pathogenesis-related protein PR1a, in leaves of plants inoculated with P. putida and REW1-I-1. Only REW1-I-1, but not P9A or P. putida induced symptoms of an hypersensitive response on tobacco leaves, bean cotyledons, and in bean suspension cultures. Phenolics and phytoalexins accumulated in bean cotyledons exposed to REW1-I-1 for 24 h but little change in levels of these compounds occurred in cotyledons inoculated with P9A and P. putida. Both suspension culture cells and roots treated with REW1-I-1 rapidly evolved more hydrogen peroxide than those exposed to P9A and P. putida. However, roots from 14-day-old plants colonized by P9A, P. putida or REW1-I-1 did not have higher levels of phenolics, phytoalexins or mRNAs for two enzymes involved in phenolic biosynthesis, phenylalanine-ammonia lyase and chalcone synthase. A selective induction of plant defense strategies upon root colonization by certain pseudomonads is apparent.     

Root Defense Responses to Fungal Pathogens: A Molecular Perspective

This review will focus on the molecular and genetic mechanisms underlying defense responses of roots to fungal pathogens. Soil-borne pathogens, including Phytophthora, Pythium, Fusarium, and Bipolaris, represent major sources of biotic stress in the rhizosphere and roots of plants. Molecular recognition and signaling leading to effective resistance has been demonstrated to occur between host and Phytophthora, or Pythium. The hypersensitive response and apoptotic cell death, two oxidative processes that limit biotrophic pathogens, generally act to exacerbate disease symptoms induced by necrotrophic organisms. Although pathogenesis-related proteins can be expressed in roots during pathogen challenge, salicylic acid has not been implicated in root-mediated interactions. Jasmonic acid and ethylene have been found to mediate parallel as well as synergistic pathways that confer partial tolerance to necrotrophic pathogens, as well as induced systemic resistance to root and foliar pathogens. Genomics approaches are revealing new networks of defense-signaling pathways, and have the potential of elucidating those pathways that are important in root-defense responses.     

Induction of Resistance in Melon to Didymella bryoniae and Sclerotinia sclerotiorum by Seed Treatments with Acibenzolar-S-methyl and Methyl Jasmonate but not with Salicylic Acid

The plant resistance activator acibenzolar‐S‐methyl (BTH), the signalling molecules salicylic acid (SA) and methyl jasmonate (MeJA) were tested by seed treatment for their ability to protect melon seedlings from gummy stem blight and white mould disease caused by the soil‐borne fungal pathogens Didymella bryoniae and Sclerotinia sclerotiorum, respectively. Didymella bryoniae infection on melon seedlings was completely suppressed by MeJA treatment. Necrotic lesions akin hypersensitive response occurred on all inoculated seedlings and prevented pathogen diffusion into healthy tissues. Didymella bryoniae infection was restricted following BTH seed treatment as well, although the percentage of necrotic lesions in comparison with the water soaked lesions was significantly lower than that from MeJA‐induced seedlings. BTH protected melon seedlings against S. sclerotiorum by the occurrence of a high percentage of necrotic lesions. A lower level of resistance was also achieved by MeJA seed treatment. The augmented level of resistance of tissues from BTH and MeJA‐treated seeds was associated with rapid increases in the activity of the pathogenesis‐related proteins chitinase and peroxidase. MeJA also determined a rapid and transient accumulation of lipoxygenase. Moreover, BTH and MeJA treatments determined the differential induction of particular de novo synthesized isoenzymes of these proteins. Results indicate that BTH and MeJA applied to melon seeds may activate on seedlings diverse metabolic pathways leading to the enhancement of resistance against distinct pathogens.     

Prodromo della Flora Umbra

Defense responses of plants are activated not only in the wounded tissues but also in the remote parts of the plants. Two different methyl jasmonate (MeJA) treatments were conducted, i.e., MeJA solution spraying of entire rosettes leaves and pasting leaf surface with lanolin squares containing MeJA. Glucosinolate profiles in leaves were similar using the two methods of MeJA treatment except for indole glucosinolates. The glucosinolate profiles in local and systemic leaves showed that the accumulation of glucosinolates in systemic leaves were delayed comparing with those in local treated leaves. Comparative proteomics were used to investigate the molecular processes underlying the glucosinolate changes in response to local and systemic MeJA induction. A total of 83 unique proteins were detected as differentially expressed between the local and systemic leaves. Functional analysis showed that redirection of metabolism from growth to defense was differentially regulated in local and systemic MeJA induction. The higher contents of indole glucosinolates in systemic leaves might arise from the induction of a long-distance signal produced in local leaves as well as from JA synthesized in systemic leaves.     

Overproduction of salicylic acid in plants by bacterial transgenes enhances pathogen resistance

After a hypersensitive response to invading pathogens, plants show elevated accumulation of salicylic acid (SA), induced expression of plant defense genes, and systemic acquired resistance (SAR) to further infection by a broad range of pathogens. There is compelling evidence that SA plays a crucial role in triggering SAR. We have transformed tobacco with two bacterial genes coding for enzymes that convert chorismate into SA by a two-step process. When the two enzymes were targeted to the chloroplasts, the transgenic (CSA, constitutive SA biosynthesis) plants showed a 500- to 1,000-fold increased accumulation of SA and SA glucoside compared to control plants. Defense genes, particularly those encoding acidic pathogenesis-related (PR) proteins, were constitutively expressed in CSA plants. This expression did not affect the plant phenotype, but the CSA plants showed a resistance to viral and fungal infection resembling SAR in nontransgenic plants.     

The effect of chemical stress on the polypeptide composition of the intercellular fluid of barley leaves

The effect of chemical stress on the polypeptide composition of the intercellular fluid of barley (Hordeum vulgare L.) and tomato (Lycopersicon esculentum Mill.) leaves has been studied. In some dicotyledonous plant species, including tomato, exposure to chemical stress leads to the denovo synthesis of intercellular proteins known as pathogenesis-related proteins which have been implicated to be part of a defence mechanism. In barley, however, no such changes in the polypeptide composition of the intercellular fluid could be detected. On the other hand, similar stress conditions induce in barley a strong accumulation of mRNA encoding leaf-specific thionins. These barley thionins represent a novel class of cell-wall proteins toxic to phytopathogenic fungi and are possibly involved in the defence mechanism. These proteins could not be detected in tomato plants. In contrast to the pathogenesis-related proteins of dicotyledonous plants, the leaf-specific thionins of barley are not present in the intercellular fluid of leaves. These results indicate that barley may have evolved a different mechanism to cope with the presence of stress.Abbreviations PAGE polyacrylamide gel electrophoresis - PR pathogenesis-related - SDS sodium dodecyl sulfate     

Reversible cytoplasmic rearrangements precede wall apposition,hypersensitive cell death and defense-related gene activation in potato/Phytophthora infestans interactions

We used video microscopy techniques as a tool for live examination of the dynamic aspects of plant/fungus interactions. Early, dynamic responses of epidermal midrib cells of leaves from a potato cultivar (Solanum tuberosum L. cv. Datura) carrying resistance gene R1 to Phytophthora infestans (race 1: compatible interaction, race 4: incompatible interaction) were monitored. Similar responses were observed in both types of interaction, ranging from no visible reaction of invaded plant cells to hypersensitive cell death. The overall defense response of each individual cell exhibited a highly dynamic behavior that appeared to be tightly coordinated with the growth of the fungus. Initial localized reactions, including major rearrangements within the cytoplasm, occurred directly at the fungal penetration site, where rapid apposition of autofluorescent material and callose took place. If fungal invasion stopped at this stage, the host cell restored its normal cytoplasmic activity and survived. Hypersensitive cell death occurred only when fungal growth had proceeded to the formation of a clearly identifiable haustorium. In such cases, cytoplasm and nucleus conglomerated around the intracellular fungal structure, followed by a sudden collapse of the whole conglomerate and an instantaneous collapse of the fungal haustorium. Only small quantitative differences between the compatible and incompatible interactions of the two fungal races were observed for these early responses of epidermal cells. In the incompatible interaction, a slightly larger number of epidermal cells responded to fungal attack. More pronounced quantitative differences between compatible and incompatible interactions occurred upon fungal invasion of the mesophyll. These differences in the number of responding cells were not reflected at the level of gene expression: the spatial and temporal activation patterns of two defense-related genes, encoding phenylalanine ammonia-lyase and pathogenesis-related protein 1, were similar in both types of interaction.Dedicated to Professor Peter Sitte, Freiburg, Germany, on the occasion of his 65th birthday     

Ultraviolet light and ozone stimulate accumulation of salicylic acid,pathogenesis-related proteins and virus resistance in tobacco

In tobacco (Nicotiana tabacum L. cv. Xanthinc), salicylic acid (SA) levels increase in leaves inoculated by necrotizing pathogens and in healthy leaves located above the inoculated site. Systemic SA increase may trigger disease resistance and synthesis of pathogenesis-related proteins (PR proteins). Here we report that ultraviolet (UV)-C light or ozone induced biochemical responses similar to those induced by necrotizing pathogens. Exposure of leaves to UV-C light or ozone resulted in a transient ninefold increase in SA compared to controls. In addition, in UV-light-irradiated plants, SA increased nearly fourfold to 0.77 g·g^–1 fresh weight in leaves that were shielded from UV light. Increased SA levels were accompanied by accumulation of an SA conjugate and by an increase in the activity of benzoic acid 2-hydroxylase which catalyzes SA biosynthesis. In irradiated and in unirradiated leaves of plants treated with UV light, as well as in plants fumigated with ozone, PR proteins 1a and 1b accumulated. This was paralleled by the appearance of induced resistance to a subsequent challenge with tobacco mosaic virus. The results suggest that UV light, ozone fumigation and tobacco mosaic virus can activate a common signal-transduction pathway that leads to SA and PR-protein accumulation and increased disease resistance.Abbreviations PR protein pathogenesis-related protein - SA salicylic acid - TMV tobacco mosaic virus - UV ultraviolet This work was financed by grants from the U.S. Department of Agriculture (Competitive Research Grants Office), Division of Energy Biosciences of U.S. Department of Energy, the Rockefeller Foundation, the New Jersey Commission for Science and Technology, and the New Jersey Agricultural Experiment Station.